mycobacterium avium subspecies paratuberculosis the cause of...

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Mycobacterium avium subspecies paratuberculosis – The cause of Crohn's disease

Ilija Barukčić

Internist: Horandstrase, DE-26441, Jever, Germany.

Email: [email protected]

Ilija Barukčić (2018) Mycobacterium avium

subspecies paratuberculosis - The cause of

Crohn's disease. viXra, 12 (3), 1-30.


Received: 2018 03, 17

Accepted: 2018 03, 17

Published: 2018 03, 17

Copyright © 2018 by Ilija Barukčić, Jever, Geramyn. All rights reserverd.

Abstract

Objective: This systematic review assesses the causal relationship between Mycobac-

terium avium subspecies paratuberculosis (MAP) and Crohn’s disease (CD).

Methods: A systematic review and meat-analysis of some impressive PCR based stud-

ies is provided aimed to answer among other questions the following question. Is there

a cause effect relationship between Mycobacterium avium subspecies paratuberculosis

and Crohn’s disease? The method of the conditio per quam relationship was used to

proof the hypothesis whether the presence of Mycobacterium avium subspecies para-

tuberculosis guarantees the presence of Crohn’s disease. In other words, if Crohn’s

disease is present, then Mycobacterium avium subspecies paratuberculosis is present

too. The mathematical formula of the causal relationship k was used to proof the hy-

pothesis, whether there is a cause effect relationship between Mycobacterium avium

subspecies paratuberculosis and Crohn’s disease. Significance was indicated by a

p-value of less than 0.05.

Result: The studies analyzed (number of cases and controls N=1076) were able to pro-

vide evidence that Mycobacterium avium subspecies paratuberculosis is a necessary

condition (a conditio sine qua non) and sufficicent conditions of Crohn’s disease. Fur-

thermore, the studies analyzed provide impressive evidence of a cause-effect relation-

ship between Mycobacterium avium subspecies paratuberculosis and Crohn’s disease.

Conclusion: Mycobacterium avium subspecies paratuberculosis is the cause of

Crohn’s disease (k=+0,377468824, p value < 0.0001).

Keywords

Mycobacterium avium subspecies paratuberculosis, Crohn’s disease, cause effect rela-

tionship, causality

1. Introduction

Crohn’s disease, described in 1904 by Polish surgeon Antoni Leśniowski [1], later in

1913 by Dalziel [2] and ultimately by Crohn, Ginzburg and Oppenheimer in 1932 [3], is a

debilitating chronic inflammatory bowel disease (IBD) of unknown cause. Leśniowski

Open Access

**Special description of the title. (dispensable)




Ilija Barukčić


-Crohn’s disease affects about 6.3 per 100,000 people-years in Europe [4]. Many times,

the initial signs and symptoms of Crohn’s disease (CD) are non-specific and can overlap

with symptoms of irritable bowel syndrome (IBS). A delay in the diagnosis of this in-

flammatory bowel disease (IBD) is associated with problems for both patients and physi-

cians. Often Crohn’s disease patients suffer from abdominal pain, malabsorption, stea-

torrhea, protein losing enteropathy, excessive diarrhea, rapid weight loss and other

symptoms which may affect their quality of life [5]. Several different extra-intestinal

complications of Crohn’s disease (events outside the gastrointestinal tract) may occur.

Lesions of Crohn’s disease begin as mucosal erosions and neutrophil infiltrates within

crypts and crypt abscesses and may progress to transmural lymphogranulomatous enteri-

tis while a cobblestoned appearance in the distal ileum and colon is observed in Crohn’s

disease patients [6]. Medical treatment of Crohn’s disease patients includes nutritional

therapy, a medication having weak anti-MAP activity with anti-inflammatory drugs, im-

munosuppressants, and sometimes antibiotics too. A view monoclonal antibodies such as

Adalimumab (Humira ®) and Infliximab (Remicade ®) are used too, to treat Crohn’s

disease patients. Most often, Crohn’s disease patients require a dangerous, costly and

time-consuming surgical intervention (laparoscopy, strictureplasty, anastomosis, bypass

surgery et cetera). A number of theories regarding the etiology of Crohn's disease are

discussed including diet, infections, other unidentified environmental factors, immune

dysregulation and autoimmune theories. Still, the cause of Crohn’s disease or some criti-

cal aspects in the pathogenesis of this disease are not known. Many authors are of the

opinion that Crohn's disease is a syndrome caused by several etiologies. Mycobacterium

avium subspecies paratuberculosis (MAP) is endemic in the bovine populations of many

countries [7] and known to be a causative agent of Johne's disease, an inflammatory

bowel disease in a variety of mammals including monkeys, chimpanzees cattle sheep,

deer, bison et cetera. Johne's disease was discovered by Dr. H.A. Johne and Dr. L. Froth-

ingham as visiting scientists from the Pathology Unit in Boston, Massachusetts at the

Veterinary Pathology Unit in Dresden by investigating the tissues of a cow from the Ol-

denburg region of Germany. The first occurrence of Johne's disease [8] in the U.S. was

published by Leonard Pearson (1868-1909) in 1908. The first description of the similari-

ties between Crohn’s disease and Johne’s disease in cattle was made in 1913 by the Scot-

tish surgeon Thomas Kennedy Dalziel [2]. The zoonotic capacities of MAP [9] and

transmission routes to humans [10], [11] have been discussed widely. Mycobacterium

avium subsp. paratuberculosis has been detected in retail cheese in about 31.7% of the

samples [12]. Due to the similarities between Johne’s disease in cattle and Crohn’s dis-

ease, it has been argued that Mycobacterium avium subspecies paratuberculosis (MAP),

which causes Johne’s disease, might also be a cause of Crohn’s disease too. Historical-

ly, MAP became the leading infectious candidate as the causative agent of Crohn’s dis-

ease. Meanwhile, the evidence to support a M. paratuberculosis infection as a cause of

Crohn's disease is mounting rapidly. Studies were able to document that up to 83% of

Crohn’s patients showed evidence of serum antibodies [13]-[18] to M. avium ss paratu-


Ilija Barukčić


berculosis. In particular, critics of the mycobacterial theory argue that MAP is not a caus-

al factor but a secondary invader [19]. The relationship between Mycobacterium avium

subspecies paratuberculosis (M. paratuberculosis) and Crohn's disease is suspected but

the evidence remains controversial.

2. Material and methods

Chronic diarrhea is the only but the most common presenting symptom of Crohn's dis-

ease. Severe disease-specific complications of this lifelong disease and a global health

problem too are common and accompanied by disabling symptoms and impaired quality

of life. The need for repeated courses of cost-expensive therapies, hospitalization and

surgery determines a substantial healthcare burden which affects the patient, the

healthcare systems and human society in general.

2.1. Search strategy

For the questions addressed in this paper, Pubmed was searched for case-control studies

conducted in any country which investigated the relationship between Mycobacterium

avium subspecies paratuberculosis (MAP) and Crohn’s disease at least by polymerase

chain reaction (PCR). The search in Pubmed was performed while using medical key

words like “case control study” and “Mycobacterium avium subspecies paratuberculosis”

and “Crohn’s disease” and “PCR DNA” et cetera. The articles found where saved as a

*.txt file while using Pubmed support (Menu: Send to, Choose Radio Button: File,

Choose Format: Abstract (text). Click buttom “create file”). The created *.txt file was

converted into a *.pdf file. The abstracts where studied within the *.pdf file. Those arti-

cles were considered for a review which provided access to data without any data access

barrier, no data access restrictions were accepted. Additionally, references from relevant

publications and review articles were checked. Case-control studies were included if they

compared the prevalence of MAP in patients with Crohn’s disease with the prevalence in

healthy controls. Studies were excluded if insufficient data were provided to calculate the

measures of relationship or if there were data access barriers.

2.2. The data of the studies analyzed

M. paratuberculosis recovers very poorly by culture from Crohn's disease tissues while

the incubation times (sometimes greater than one year) can be extremely long. In this

context, novel laboratory techniques [20] (Southern Blot hybridization, Immunohisto-

chemistry (IHC), introduced by Coons [21] in 1941, In-situ hybridization (ISH), de-

scribed in the year 1969 by Joseph G. Gall [22], Fluorescent ISH (FISH), RNA in situ

hybridization (RNA ISH), Polymerase chain reaction (PCR), Nested PCR, Quantitative

polymerase chain reaction (QPCR) et cetera) can improve our understanding of the path-


Ilija Barukčić


ogenesis of Crohn’s diseases. The data of the studies [23]-[39] analyzed, are presented by

the 2 by 2-table (Table 1). The meaning of the abbreviations at, bt, ct, dt, Nt of table 1

(Table 1), table 2 (Table 2), table 3 (Table 3), table 4 (Table4) are explained by a 2 by

2-table (Table 5).

Table 1. The data of the studies considered for a meta-analysis.

Mycobacterium avium subspecies paratuberculosis

Crohn's Disease Healthy control Sample Size

Reviiew MAP + MAP - MAP + MAP -

Study Id at ct bt dt at+bt+ct+dt=Nt

Sanderson et al. [23] 26 14 5 35 80

Fidler et al. [24] 4 27 0 30 61

Hulten et al. [25] 7 30 0 22 59

Ryan et al. [26] 6 9 0 12 27

Bull et al. [27] 34 3 9 25 71

Autschbach et al. [28] 22 14 4 73 113

Sechi et al. [29] 30 5 3 26 64

Romero et al. [30] 10 2 1 5 18

Szkaradkiewicz et al. [31] 10 6 1 11 28

Kirkwood et al. [32] 22 34 6 33 95

Mendoza et al. [33] 30 0 0 10 40

Tuci et al. [34] 21 10 11 21 63

Lee et al. [35] 5 14 0 19 38

Nazareth et al. [36] 27 13 11 18 69

Timms et al. [37] 6 15 0 21 42

Khan et al. [38] 16 53 3 46 118

Zamani et al. [39] 18 10 6 56 90

Total events 294 259 60 463 1076

2.3. Statistical analysis

All statistical analyses were performed with Microsoft Excel version 14.0.7166.5000

(32-Bit) software (Microsoft GmbH, Munich, Germany). In order to simplify the under-

standing of this article, to increase the transparency for the reader and to correct some of

the misprints of former publications, several of the following lines are repeated word by

word and taken from former publications.


Ilija Barukčić


2.3.1. The 2x2 table

The 2x2 table in this article is defined [40]-[62] in general more precisely (Table 5) as

follows.

Table 5. The sample space of a contingency table

Conditioned Bt

(Crohn’s disease)

Yes = +1 Not = +0 Total

Condition At

(MAP PCR DNA)

Yes =+1 at bt At

Not = +0 ct dt At

Total Bt Bt Nt

In general it is (a+b) = At, (c+d) = At, (a+c) = Bt, (b+d) = Bt and at+bt+ct+dt=Nt. Equally,

it is Bt+Bt = At + At = Nt. In this context, it is p(at)=p(At Bt), p(At) = p(at)+p(bt) or in

other words p(At)= p(At Bt)+p(At Bt) while p(At) is not defined as p(at). In the same

context, it should be considered that p(Bt) = p(at)+p(ct) = p(At Bt) +p(ct) and equally

that p(Bt) = 1- p(Bt) =p(bt)+p(dt). In point of fact, the joint probability of At and Bt is de-

noted by p(At Bt). It is p(at)+p(ct)+p(bt)+p(dt) = 1. These relationships are viewed by

the table (Table 6) as follows.

Table 6. The probabitlities of a contingency table

Conditioned

Bt

Yes = +1 No = +0 Total

Condition

At

Yes =+1 p(at) = p(At Bt) p(bt) p(At)

No = +0 p(ct) p(dt) p(At)

Total p(Bt) p(Bt) 1

2.3.2. Independence

In the case of independence of At and Bt it is

(1)

2.3.3. Sufficient condition (conditio per quam; material conditional)

The mathematical formula of the sufficient condition relationship (conditio per quam)

[40]-[62] of a population was defined as

(2)

t t t

t t

t

a c dp A B 1

N

t t t tp A B p A p B


Ilija Barukčić


and used to proof the hypothesis: if At then Bt . In particular it is

(3)

Scholium.

Although the work on study bias is vast and therefore quite difficult to survey adequately,

we can at least point out that several factors including the study design can have an im-

pact on bias with respect to the sufficient condition too. The question is, what is the rela-

tionship between the independence of an event At (a condition) and another event Bt

(conditioned) and the sufficient condition relationship. Especially, is it possible that an

event At is a sufficient condition of an event Bt even if event At (a sufficient condition) is

independent of an event Bt (the conditioned). In this context, the conditio per quam was

defined as

(4)

or a

(5)

Under conditions where an event At is independent of an even Bt it is equally true that

(6)

Substituting this relationship into the equation before and rearranging equation it is

(7)

or

(8)

or

(9)

Only under conditions where p(Bt) = 1, it is possible that At as a sufficient condition of Bt

even if At is independent of Bt and vice versa, otherwise not. In other words, a statistical-

ly significant conditio per quam relationship is very convincing if at the same time an

t t t t t

tt t t t

t t t t t

t t

p A B p a p c p d 1

p A B p A B p A 1

p A B p A B 1 p A 1

p A B 1

tt t t tp A B p A B p A 1

t t t t tp A B p A B 1 p A 1

t t t tp A B p A p B

t t tp A p B 1 p A 1

t t tp A p B p A

tp B 1.


Ilija Barukčić


event At is not independent of and event Bt and vice versa. Thus far, an inappropriate

study design and other sources of possible bias, diminish in their importance if a statisti-

cal significant conditio per quam relationship is supported by the absence of independ-

ence of the same two events.

2.2.3. Necessary condition (conditio sine qua non)

The formula of the necessary condition (conditio sine qua non) [40]-[62] relationship was

derived as

(10)

and used to proof the hypothesis: without At no Bt.

2.2.4. Necessary and sufficient condition (material biconditional)

The necessary and sufficient condition relationship was defined [40]-[62] as

(11)

2.3.4. The X² goodness of fit test of a necessary condition

Under conditions where the chi-square [63] goodness of fit test cannot be used it is possi-

ble to use an approximate and conservative (one sided) confidence interval as discussed

by Rumke [64], Louis [65], Hanley et al. [66] and Jovanovic [67] known as the rule of

three. Under some circumstances, the rule three and other methods can be used to test the

significance of a necessary condition. In this publication, the chi-square goodness of fit

test was used to determine whether sample data are consistent with a hypothesized (theo-

retical) distribution of a necessary condition. In particular, the hypotheses can take the

following form.

H0: The sample distribution do agree with the hypothetical (theoretical) distribution of a

necessary condition.

HA: The sample distribution do not agree with the hypothetical (theoretical) distribution

of a necessary condition.

The X² Goodness-of-Fit Test can be shown schematically as

(12)

The degrees of freedom are calculated as N-1. Interestingly, if there is no discrepancy

2

t Nt t2

t 1 t

Observed Expected

Expected

t t ttt t t t

a b dp A B p A B p B 1

N

t tt tt t t t

a dp A B p A B p A B 1

N


Ilija Barukčić


between an observed and a theoretical distribution at all, then the value of the calculated

X²=0. As the discrepancy between an observed and the theoretical distribution of a nec-

essary condition becomes larger, the X² becomes larger. This X² values are evaluated by

the known X² distribution. An adjustment (Yate’s correction for continuity) can be used

when there is one degree of freedom. When there is more than one degree of freedom, the

same adjustment is not used. Applying this to the formula above, we find the X² Good-

ness-of-Fit Test with continuity correction shown schematically as

(13)

Under circumstances, where the term (|Observedt - Expectedt|) is less than ½, the continu-

ity correction should be omitted. The theoretical (hypothetical) distribution of a necessary

condition is shown schematically by the 2x2 table (Table 7).

Table 7. The theoretical distribution of a necessary condition (conditio sine qua non).

Conditioned Bt

Yes = +1 No = +0 Total

Condition

At

Yes =+1 at bt (at+bt)

No = +0 ct=0 dt (ct+dt)

Total (at+ct) (bt+dt) (at+bt+ct+dt)

The theoretical distribution of a necessary condition (conditio sine qua non) is determined

by the fact that c=0. The X² Goodness-of-Fit Test with continuity correction of a neces-

sary condition (conditio sine qua non) is calculated as

(14)

or more simplified as

(15)

Under these circumstances, the degree of freedom is d.f. = N-1=2-1=1. The conditio

sine qua non model can be used widely and is one of the new and appropriate methods of

2

t tt N2

t 1 t

1Observed Expected

2

Expected

2 2 2

t t t t t t t t t t

2

t t t t t t

1 1 1a b a b d c d d c d

2 2 2SINE 0

a b c d c d

2

t

2

t t

1c

2SINE 0

c d


Ilija Barukčić


analysis of binary outcome variables. In this context, meta-analysis and systematic re-

views aims to combine effects estimated from several studies to achieve greater precision

of the conclusions drawn and can provide us with more convincing and reliable evidence

of some special aspects of medicine. In meta-analysis the heterogeneity between the

studies can be modelled via the additive properties of the chi square distribution too. In

general, let Xt denote n independent random variables which follow a chi-square distribu-

tion. The sum of these independent chi-square variate is itself a chi-square variate which

is known as the additive property of independent chi-squares. There may be disad-

vantages in the use of the chi-square-goodness-of-fit test. Still, the chi square distribution,

a continuous probability distribution, is related to the standard normal distribution and is

a simple and good measure of model adequacy. However, a particular concern with the

use of the chi-square-goodness-of-fit test is a priori justified if expected cell frequencies

of a 2x2 table are too small (all are less than one).

2.3.5. The mathematical formula of the causal relationship k

Huxley [68] and Darwin [69] claimed more than a century ago that humans share recent

common ancestors with the African apes. Modern molecular methods have spectacularly

confirmed their prediction. Genomic divergences between humans and other hominoids

and especially our closest living evolutionary relatives the common chimpanzee (Pan

troglodytes) and bonobo (Pan paniscus or pygmy chimpanzee) are very small but not ze-

ro. Ebersberger et al. [70], Fujiyama et al. [71] and other sequenced the chimpanzee ge-

nome. According to Ebersberger et al. “the chimpanzee genome were sequenced and

compared to corresponding human DNA sequences ... the average sequence difference is

low (1.24%)” [70]. The Chimpanzee Sequencing and Analysis Consortium calculated

“the genome-wide nucleotide divergence between human and chimpanzee to be 1.23%”

[72] and confirmed results from other and more limited studies. In other words, the dif-

ference between chimpanzee genome and compared to corresponding human DNA se-

quences is very small. Still there is a difference and this very small difference makes the

difference. A chimpanzee is not a human being, a human being is not a chimpanzee. Even

if both are similar and “relatives” both are equally not the same. The relationship between

the mathematical formula of the causal relationship k [40]-[62] and the closest existing

mathematical relatives, Pearson's measures of relationships, is similar to the circum-

stances aforementioned. In contrast to Pearson's product-moment correlation coefficient

[73] or to Pearson's Phi [74] Coefficient (Mean Square Contingency Coefficient et cetera,

the mathematical formula of the causal relationship k [40]-[62] is defined at every single

event, at every single Bernoulli trial t, as

(16)

R t 0 t R t 0 t

R t 0 t2

R t 0 tR t 0

p U W p U p Wk U , W

p U p U p W p W


Ilija Barukčić


where RUt denotes the cause and 0Wt denotes the effect while the chi-square distribution

[63] can be applied to determine the significance of causal relationship k. This small dif-

ference makes the difference. Only under conditions where the probability of events is

constant from trial to trial, we can extrapolate from one Bernoulli trial to N Bernoulli tri-

als with some consequences one of which is that

(17)

or that

(18)

or at the end

(19)

where N is the sample size, at=Ntp(RUt 0Wt), RUt = Np(RUt), RUt = Ntp(RUt),

0Wt=Ntp(0Wt), 0Wt=Ntp(0Wt). Several factors can have an impact on the calculated

causal relationship k with the potential of bias.

Scholium.

Firstly, the relationship between condition and cause has an impact on the causal rela-

tionship k. A proper and deeper analysis of the relationship between cause and condition

is beyond the scope of this article and can be found in literature [40]-[62]. We will be

concerned with the latter sort of entity in this article from a pragmatically point of view.

In the hope of casting light on the tricky problems of the relationship between condition

and cause, the concept of independence is of use too. The question whether an event At

can be a (necessary, sufficient, necessary and sufficient) condition of an event Bt even if

both are independent of each other, is already answered few lines before. Still, under

which circumstances can we treat an event as a cause or as the cause of another event?

Can an event be a cause of another event without being a (necessary, sufficient, necessary

and sufficient et cetera) condition of the same event? The concept of this article is re-

stricted on its capacity to bring high degrees of conceptual exactness and rigour to ques-

tions like these but not incapable. Most authors who have written on the question of the

relationship between condition and cause came to different conclusions. Currently still

worthy of consideration is the remark of von Bar.

“Die erste Voraussetzung, welche erforderlich ist, damit eine Erscheinung als die Ursache

einer anderen bezeichnet werden könne, ist, daß jene eine der Bedingungen dieser sein.

t t R t 0 t

R t 0 t2

R t 0 tR t 0

N a U Wk U , W

U U W W

t t R t 0 t R t 0 t

R t 0 t2

Rt tt t 0R t 0

N N p U W p U p Wk U , W

N N p U p U p W p W

R t 0 t R t 0 t

R t 0 t2

R t

t t t t

t t t t 0 tR t 0

N N p U W N p U N p Wk U , W

N p U N p U N p W N p W


Ilija Barukčić


Würde die zweite Erscheinung auch dann eingetreten sein, wenn die erste nicht vorhan-

den war, so ist sie in keinem Falle Bedingung und noch weniger Ursache. Wo immer eine

Kausalzusammenhang behauptet wird, da muß er wenigstens diese Probe aushalten. …

Jede Ursache ist nothwendig auch eine Bedingung eines Ereignisses; aber nicht jede Be-

dingung ist Ursache zu nennen. “ [75]

Translated into English:

‘The first requirement, which is required, thus that something could be called as the cause

of another, is that the one has to be one of the conditions of the other. If the second

something had occurred even if the first one did not exist, so it is by no means a condition

and still less a cause. Wherever a causal relationship is claimed, the same must at least

withstand this test. ... Every cause is necessarily also a condition of an event too; but not

every condition is cause too.‘

A cause is a condition of an event too but not necessarily vice versa. A condition of an

event must not be equally the cause of the same event. Thus far, a study which provides

evidence of a significant causal relationship k without at the same time providing evi-

dence of a significant necessary condition, or of a significant sufficient condition or of a

significant necessary and sufficient condition should be treated with some cautious.

2.3.6. The chi square distribution

The chi-squared distribution [63] is a widely known distribution and used in hypothesis

testing, in inferential statistics or in construction of confidence intervals. The critical val-

ues of the chi square distribution are visualized by Table 8.

Table 8. The critical values of the chi square distribution (degrees of freedom: 1).

p-Value One sided X² Two sided X²

The chi square

distribution

0,1000000000

0,0500000000

0,0400000000

0,0300000000

0,0200000000

0,0100000000

0,0010000000

0,0001000000

0,0000100000

0,0000010000

0,0000001000

0,0000000100

0,0000000010

0,0000000001

1,642374415

2,705543454

3,06490172

3,537384596

4,217884588

5,411894431

9,549535706

13,83108362

18,18929348

22,59504266

27,03311129

31,49455797

35,97368894

40,46665791

2,705543454

3,841458821

4,217884588

4,709292247

5,411894431

6,634896601

10,82756617

15,13670523

19,51142096

23,92812698

28,37398736

32,84125335

37,32489311

41,82145620


Ilija Barukčić


2.3.7. The X² goodness of fit test of a causal relationship k

Under some circumstances the chi-square [48] goodness of fit test can be used to test the

significance of a causal relationship. Under conditions where the probability of events is

constant from trial to trial, we expect a constant causal relationship kt. In other words, at

each Bernoulli trial t it is

(20)

where pk(RUt,0Wt) denotes the causal relationship within the population. Performing N

Bernoulli trials (Sample size N), the basic relationship will not change. It follows that

(21)

(assumed that |pk(RUt,0Wt)=1|) or that

(22)

Simplifying equation we obtain

(23)

Multiplying equation by itself it is

(24)

or

(25)

Dividing equation by N*(|pk(RUt,0Wt)=1|)=N, we obtain

(26)

or

(27)

or the X² value as

(28)

The chi square (X2) statistic can be used to investigate whether the observed distribution

of the causal relationship differ from the theoretical expected distribution of the causal

relationship. The table 8 (Table 8) contains the critical values of the chi-square distribu-

tion (degrees of freedom, df =1). Upper-tail and lower-tail critical values of the

chi-square distribution with v degrees of freedom are provided by software packages.

R t 0 t p R t 0 tk U , W k U , W 1

R t 0 tN k U , W N 1

R t 0 tN k U , W N 1 0

R t 0 tN k U , W 1 0

R t 0 t R t 0 tN k U , W 1 N k U , W 1 0 0

2

2

R t 0 tN k U , W 1 0

2

2

R t 0 tN k U , W 1 00

N N

2

R t 0 tN k U , W 1 0

2

2

R t 0 tN k U , W 1 0


Ilija Barukčić


3. Results

3.1. Without the presence of Mycobacterium avium subspecies paratu-berculosis no presence of Crohn's disease

Claims.

Null hypothesis:

The presence of Mycobacterium avium subspecies paratuberculosis is a necessary condi-

tion (a conditio sine qua non) of Crohn's disease. In other words, the sample distribution

agrees with the hypothetical (theoretical) distribution of a necessary condition.

Alternative hypothesis:

The presence of Mycobacterium avium subspecies paratuberculosis is not a necessary

condition (a conditio sine qua non) of Crohn's disease. In other words, the sample distribu-

tion does not agree with the hypothetical (theoretical) distribution of a necessary condition.

The significance level (Alpha) below which the null hypothesis will be rejected is al-

pha=0,05.

Proof.

The data reviewed by this article which investigated the relationship between the presence

of Mycobacterium avium subspecies paratuberculosis and Crohn's disease are viewed by

the table (Table 2). Altogether, 17 studies were meta-analyzed while the level of signifi-

cance was alpha = 0,05. Altogether, 10 from 17 studies provide significant evidence of a

conditio sine qua non relationship between Mycobacterium avium subspecies paratuber-

culosis and Crohn's disease. The sample size of the study of Romero et al. [24] was small

and has been analyzed and is significant according to the Rule of three. In the same re-

spect, 17/17 studies analysed provided evidence of a significant cause effect relationship

between Mycobacterium avium subspecies paratuberculosis and Crohn's disease. In other

words, without a Mycobacterium avium subspecies paratuberculosis infection of human

intestinal tract no Crohn's disease. Due to methodological inconsistencies, 7 of 17 studies

reanalyzed failed to provide statistically significant evidence of this relationship. In point

of fact, the presence of Mycobacterium avium subspecies paratuberculosis inside human

intestinal tract is a necessary condition (a conditio sine qua non) of Crohn's disease. In

other words, without the presence of Mycobacterium avium subspecies paratuberculosis

inside human intestinal tract no Crohn's disease.

Q. e. d.


Ilija Barukčić


Table 2. Without MAP infection of human intestinal tract no Crohn’s disease.

Author Year Country at bt ct dt at+bt+dt Nt (at+bt+dt)/Nt X²(Sine) k p val (k)

Mendoza et al. [33] 2010 Spain 30 0 0 10 40 40 1 0,025 1 2,53963E-10

Bull et al. [27] 2003 UK 34 9 3 25 68 71 0,957746479 0,2232142 0,6687230 1,75302E-0

Romero et al. [30] 2005 USA 10 1 2 5 16 18 0,888888889 Rule of 3 0,6446583 0,00623700

Sechi et al. [29] 2005 Italy 30 3 5 26 59 64 0,921875 0,6532258 0,7507418 1,90233E-0

Zamani et al. [39] 2017 Iran 18 6 10 56 80 90 0,888888889 1,3674242 0,5716838 5,84534E-0

Szkaradkiewicz et al. [31] 2007 Poland 10 1 6 11 22 28 0,785714286 1,7794117 0,5488604 0,00368079

Autschbach et al. [28] 2005 Germany 22 4 14 73 99 113 0,876106195 2,0948275 0,6189973 4,70347E-1

Tuci et al. [34] 2011 Italy 21 11 10 21 53 63 0,841269841 2,9112903 0,3336693 0,00808693

Ryan et al. [26] 2002 Ireland 6 0 9 12 18 27 0,666666667 3,4404761 0,4780914 0,01298297

Sanderson et al. [23] 1992 UK 26 5 14 35 66 80 0,825 3,7193877 0,53881591 1,44051E-0

Total

207 40 73 274 521 743 0,701211306 16,21425795 0,4124364

Alpha = 0,05

Degrees of freedom = 9 Degr. of fr. = 1

X² (Critical) SINE = 16,9189776 Chi crit. (k) = 3,841458821

X² (Calculatedl) SINE= 16,5356865 X² calc. (k)= 126,3870966

k= 0,41243638


Ilija Barukčić


3.2. If presence of Mycobacterium avium subspecies paratuberculosis then presence of Crohn's disease

Claims.

Null hypothesis:

The presence of Mycobacterium avium subspecies paratuberculosis is a sufficient condi-

tion (a conditio per quam) of Crohn's disease. In other words, the sample distribution

agrees with the hypothetical (theoretical) distribution of a sufficient condition.


The presence of Mycobacterium avium subspecies paratuberculosis is not a sufficient con-

dition (a conditio per quam) of Crohn's disease. In other words, the sample distribution

does not agree with the hypothetical (theoretical) distribution of a sufficient condition.


pha=0,05.

Proof.


of Mycobacterium avium subspecies paratuberculosis inside human intestinal tract and

Crohn's disease are viewed by the table (Table 3). Altogether, 17 studies were me-

ta-analyzed with n=1076 number of cases and controls while the level of significance was

alpha = 0,05. Thus far, 17 out of 17 studies provided significant evidence of a conditio per

quam relationship between Mycobacterium avium subspecies paratuberculosis and Crohn's

disease. The studies of Ryan et al. [26], Romero et al. [30] and Szkaradkiewicz et al. [31]

were analysed and are significant according to the Rule of three. In the same respect, 17/17

studies analysed provided evidence of a significant cause effect relationship between My-

cobacterium avium subspecies paratuberculosis and Crohn's disease. In other words, if in-

fection of human intestinal tract by Mycobacterium avium subspecies paratuberculosis

then Crohn's disease. In point of fact, the presence of Mycobacterium avium subspecies

paratuberculosis within human intestinal tract is a sufficient condition (a conditio per

quam) of Crohn's disease. In other words, if presence of Mycobacterium avium subspecies

paratuberculosis within human intestinal tract then presence of Crohn's disease.

Q. e. d.


Ilija Barukčić


Table 3. If MAP infection then CD.

Author Year Country at bt ct dt at+ct+dt Nt (at+tc+dt)/Nt X²(IMP) k p val (k)


Romero et al. [30] 2005 USA 10 1 2 5 17 18 0,94444444 Rule of 3 0,64465837 0,00623700 Szkaradkiewicz et al. [31] 2007 Poland 10 1 6 11 27 28 0,96428571 Rule of 3 0,54886043 0,00368079

Hulten et al. [25] 2001 USA 7 0 30 22 59 59 1 0,03571429 0,28291621 0,02977124

Ryan et al. [26] 2002 Ireland 6 0 9 12 27 27 1 Rule of 3 0,47809144 0,01298297

Timms et al. [37] 2016 Australia 6 0 15 21 42 42 1 0,04166667 0,40824829 0,00815097

Lee et al. [35] 2011 Canada 5 0 14 19 38 38 1 0,05 0,38924947 0,01641770

Fidler et al. [24] 1994 UK 4 0 27 30 61 61 1 0,0625 0,26059876 0,04181653


Khan et al. [38] 2016 India 16 3 53 46 115 118 0,97457627 0,32894737 0,22880148 0,01293985


Sanderson et al. [23] 1992 UK 26 5 14 35 75 80 0,9375 0,65322581 0,53881591 1,44051E-06

Kirkwood et al. [32] 2009 Australia 22 6 34 33 89 95 0,93684211 1,08035714 0,257886 0,01195188


Bull et al. [27] 2003 UK 34 9 3 25 62 71 0,87323944 1,68023256 0,66872302 1,75302E-08

Nazareth et al. [36] 2015 Portugal 27 11 13 18 58 69 0,84057971 2,90131579 0,29342187 0,01479555

Tuci et al. [34] 2011 Italy 21 11 10 21 52 63 0,82539683 3,4453125 0,33366935 0,00808693

Total

294 60 259 463 1016 1076 0,94423792 12,2085699 0,3774688

Alpha = 0,05


X² (Critical) IMP = 23,6847913 Chi crit. k = 3,84145882

X² (Calculatedl) IMP= 12,2085699 X² calc. (k)= 153,311399

k= 0,377468824


Ilija Barukčić


3.3. Mycobacterium avium subspecies paratuberculosis is neces-sary and sufficient condition of Crohn's disease

Claims.

Null hypothesis:

The presence of Mycobacterium avium subspecies paratuberculosis is a necessary and suf-

ficient condition of Crohn's disease. In other words, the sample distribution agrees with the

hypothetical (theoretical) distribution of a necessary and sufficient condition.


The presence of Mycobacterium avium subspecies paratuberculosis is not a necessary and

sufficient condition of Crohn's disease. In other words, the sample distribution does not

agree with the hypothetical (theoretical) distribution of a necessary and sufficient condi-

tion.


pha=0,05.

Proof.


of Mycobacterium avium subspecies paratuberculosis and Crohn's disease are viewed by

the table (Table 4). Altogether, in this proof, 17 studies were meta-analyzed while the lev-

el of significance was alpha = 0,05. In toto, 8 studies were able to provide significant evi-

dence that Mycobacterium avium subspecies paratuberculosis is a necessary and sufficient

condition of Crohn's disease. The study of Romero et al. [30] have been analysed and was

significant according to the Rule of three. In point of fact, more or less older studies failed

to provide evidence of this relationship. In the same respect, 17/17 studies analyzed pro-

vided evidence of a significant cause effect relationship between Mycobacterium avium

subspecies paratuberculosis and Crohn's disease and thus far the absence of independence.

In other words it is true that without the presence of Mycobacterium avium subspecies

paratuberculosis within human intestinal tract no presence of Crohn's disease and at the

same time it is equally true that if presence of Mycobacterium avium subspecies paratu-

berculosis in human intestinal tract then presence of Crohn's disease too. In particular,

Mycobacterium avium subspecies paratuberculosis is a necessary and sufficient condition

of Crohn's disease.

Q. e. d.


Ilija Barukčić


Table 4. MAP infection of human intestinal tract is a necessary and sufficient condition of CD.

Author Year Country at bt ct dt at+dt at+bt+ct+dt=Nt (at+dt)/Nt X²(Sine and Imp) k p val (k)


Romero et al. [30] 2005 USA 10 1 2 5 15 18 0,8333333 Rule of 3 0,64465837 0,006237007


Szkaradkiewicz et al. [31] 2007 Poland 10 1 6 11 21 28 0,75 1,802139037 0,54886043 0,003680795

Bull et al. [27] 2003 UK 34 9 3 25 59 71 0,8309859 1,903446844 0,66872302 1,75302E-08



Ryan et al. [26] 2002 Ireland 6 0 9 12 18 27 0,6666667 3,482142857 0,47809144 0,012982973

Total

160 24 49 218 378 451 0,8381375 13,25750416 0,6437068

Alpha = 0,05


X² (Critical) = 14,06714045 Chi crit. k = 3,841458821

X² (Calculated) = 13,25750416 X² calc. (k)= 186,875687

k= 0,643706849


Ilija Barukčić


3.4. Mycobacterium avium subspecies paratuberculosis is the cause of Crohn's disease

Claims.

Null hypothesis: (no causal relationship)

There is no significant causal relationship between an infection by Mycobacterium avium

subspecies paratuberculosis and Crohn's disease.

(k=0).

Alternative hypothesis: (causal relationship)

There is a significant causal relationship between an infection by Mycobacterium avium

subspecies paratuberculosis and Crohn's disease.

(k0).

Conditions.

Alpha level = 5%.

The two tailed critical Chi square value (degrees of freedom = 1) for alpha level 5% is

3.841458821.

Proof.

The data for this hypothesis test were provided by different studies and are illustrated by a

table (Table 3). The causal relationship k(Mycobacterium avium subspecies paratubercu-

losis, Crohn's disease) is calculated according to [40]-[62]. Again, 17 studies were me-

ta-analyzed with n=1076 number of cases and controls while the level of significance was

alpha = 0,05. Thus far, all the 17 studies analyzed provided evidence of a significant cause

effect relationship between Mycobacterium avium subspecies paratuberculosis and

Crohn's disease. In other words, Mycobacterium avium subspecies paratuberculosis and

Crohn's disease are not only not independent of each other. Besides of the methodological

difficulties associated with the studies analyzed the majority of the studies provided sig-

nificant evidence of a necessary condition, of a sufficient condition and of a necessary and

sufficient condition. The conclusion appears to be inescapable: Mycobacterium avium

subspecies paratuberculosis is the cause of Crohn's disease (k=+0,377468824, p value <

0.0001)..

Q. e. d.


Ilija Barukčić


3.5. Antibiotic therapy and Crohn's disease

One of the most convincing contributions to the evidence of a role for MAP in Crohn’s

disease would be a proof that a treatment with appropriate antibiotics for sufficient dura-

tion able kill the organism leads to remission of Crohn’s disease. In this context, different

antibiotic drug combinations, called anti-MAP regimen, including clarithromycin (CLA),

rifabutin (RIF) and Clofazimine (CLO) investigated in multiple randomized clinical trials

have shown [76]-[80] promising results. A complete healing of ulcers in Crohn’s disease

patients after more than 6 months of treatment with a rifabutin (RIF) and clarithromycin

(CLA) regimen, an inhibitor of CYP34A [81], has been reported by several studies. A

2007 case study reported that one Crohn's disease patient who was being treated with an-

ti-MAP therapy drugs, attained complete clinical remission [82]. Selby et al. [83] con-

ducted a prospective, parallel, placebo-controlled, double-blind, randomized treatment trial

using the combination of clarithromycin, rifabutin, and clofazimine. Two hundred thirteen

patients were randomized while the study design of the study of Selby et al. [83]included

an initial 16-week phase in which all patients received prednisolone in addition to trial

medications. Remission was defined as Crohn’s Disease Activity Index < 150. Using

combination antibiotic therapy with rifabutin, clarithromycin, and clofazimine Selby et al.

[83] did not find evidence of a sustained benefit for Crohn’s disease patient. At the end of

week 16 period, there was only “a significantly greater percentage of subjects in remission

in the antibiotic arm (67/102 [66%]) than in the placebo arm (55/111 [50%]) (P = .02)”.

Selby et al. [83] concluded that their finding does not support a significant contribution of

Mycobacterium avium subspecies paratuberculosis in the pathogenesis of Crohn’s disease.

Thus, what unifies the conclusions of the abovementioned study group [83] is seemingly

the disparate misuse of statistical methods and the data of their study made publicly avail-

able for further investigations to an extent which demands and justifies several critical re-

marks. Firstly. The inappropriate definition of remission of Crohn’s disease (defined as

Crohn’s Disease Activity Index < 150) is highly subjective, unfair and has underestimated

Crohn’s Disease cases achieved remission. The use of Crohn’s Disease Activity Index has

overestimated the number of cases in the placebo group which achieved remission. Thera-

peutic monitoring can and should be grounded on objective parameters too like C-reactive

protein, Erythrocyte sedimentation rate, Procalcitonin (PCT), antibody (Ab) against My-

cobacterium avium ssp. Paratuberculosis, faecal calprotectin, colonoscopy and other. Sec-

ondly. The abovementioned study presented contradictory outcomes while at the same

time it cannot be excluded that subjects were not exposed to the correct dose of drugs.

Clofazimine capsules were reencapsulated and it was found that the clofazimine capsules

did not rupture in vitro. “Because of this, there was a period of approximately 10 months

during which it was possible that subjects were not exposed to the correct dose of clo-

fazimine.” [83] In contrast to Selby et al. [83], Gui et al. [84] treated 46 patients with se-

vere Crohn's disease with rifabutin in combination with a macrolide antibiotic (clarithro-

mycin or azithromycin). An improvement in inflammatory parameters was observed and

the clinical remission based on the Harvey–Bradshaw activity index was induced in 43


Ilija Barukčić


(93.5%) patients. The studies concerning chemotherapy with antimycobacterial agents

have given mixed or contradictory [85] results in Crohn’s disease patients. Thus far, a

causal relationship between M. paratuberculosis and Crohn’s disease derived from chem-

otherapy studies has not been demonstrated beyond any reasonable doubt.

4. Discussion

Other systematic reviews and meta-analysis assessed already the evidence for an associa-

tion between Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn's dis-

ease (CD). Does another review make sense at all? Feller et al. [86] were not able to solve

the problem and concluded that the role of Mycobacterium avium subspecies paratubercu-

losis (MAP) in the aetiology of Crohn's disease remains to be defined. The systematic re-

view and meta-analysis of the Mycobacterium avium subspecies paratuberculosis and

Crohn's disease published by Abubakar et al. [87] (UK) reviewed 47 studies demonstrated

that the relationship between MAP and CD “remains controversial and inconclusive. Fu-

ture studies should determine whether there is a pathogenic role.” Mycobacterium avium

subspecies paratuberculosis is a robust and phenotypically versatile pathogen which is

widespread in domestic livestock, present in retail pasteurized cows' milk and potentially,

elsewhere. A high-temperature short-time pasteurization of milk appears not effectively to

kill M. paratuberculosis in milk [88]. Even water supplies are also at risk. Mycobacterium

avium subspecies paratuberculosis (MAP) has the potential to cause chronic inflammation

of the intestine in many species, including primates. Crohn's disease has long been sus-

pected of having a mycobacterial cause. Still, for many years the role of mycobacteria,

specifically Mycobacterium paratuberculosis in Crohn's disease has aroused considerable

controversy. The inconsistencies throughout the studies on the relationship between My-

cobacterium paratuberculosis and Crohn's disease are very great and due to several factors.

First of all, it is known that Mycobacterium paratuberculosis is very difficult to be detected

by culture and due to the heterogeneous nature of CD the rate of detection of M. avium

subsp. paratuberculosis in individuals with CD can vary extremely. The development of

highly sensitive and M. paratuberculosis-specific polymerase chain reaction assays and

appropriate in situ hybridization methods is highly welcomed. Further technical and

methodological advances should allow the identification and/or isolation of M. paratuber-

culosis from a significantly higher proportion of Crohn's disease tissues compared too

controls. Clinical differences in patients studied, including treatment regimens and the du-

ration of disease are not considered in an appropriate manner. Patients studied were ana-

lyzed by different methods while the sensitivity of the methods used was not determined.

In addition to these inconsistencies, the methodological uncertainty surrounding the role of

Mycobacterium avium subsp paratuberculosis (Map) in Crohn's disease is great. The stud-

ies analyzed are full of contradictory findings due to small sample size, the lack of uni-

formity in the materials and methods used by many authors and other factors too. Under

such circumstances, is it possible at all to provide new and convincing perspectives [89]?

One might argue that in line with problems like these, the question is justified whether a


Ilija Barukčić


decision about a relationship between M. avium subsp. paratuberculosis and Crohn's dis-

ease under such conditions is possible at all. Still, the extent to which several studies were

able to determine a strong and significant relationship between Mycobacterium avium

subsp. paratuberculosis and Crohn's disease reduces the penumbra of uncertainty sur-

rounding the methodological problems of the studies dramatically.

In toto, 10 of 17 studies analysed using the ultra sensitive polymerase chain reaction

(PCR) for the detection of M. avium subsp. paratuberculosis within Crohn's disease pro-

vided convincing evidence of a conditio sine qua non relationship between M. avium

subsp. paratuberculosis and Crohn's disease patients. In other words, without M. avium

subsp. paratuberculosis no Crohn's disease. The involvement of M. avium subsp. para-

tuberculosis in Crohn's disease (CD) in humans has been uncertain because of several sub-

stantial difficulties especially in detecting this pathogen. Still, even if 7 studies failed on

the point aformentioned, the question is how could 10 studies document such an evidence.

At the same time, M. avium subsp. paratuberculosis and Crohn's disease are causally re-

lated.

All studies analyzed provide convincing evidence that the presence of M. avium subsp.

paratuberculosis gurantees the presence of Crohn's disease. In other words, if an infection

with M. avium subsp. paratuberculosis is present, then Crohn's disease is present too (X²

Calculated = 12,2085699 and is less than X² Critical = 23,6847913, degrees of freedom = 14). At

the same time, M. avium subsp. paratuberculosis and Crohn's disease were not independ-

ent of each other. Necessary and sufficient conditions are converses of one another. If it is

true that if MAP, then CD, the converse is automatically true too: without CD no MAP,

the data presented support such a hypothesis too. In principle, inflammatory bowel disease

can arise when the mucosal barrier is compromised in its defense against challenges from

the intestinal microbiota by own immune system or by other factors with the consequence

that a MAP infection is only a secondary bacterial infection at an immunocompromised

site. Theoretically, such a conclusion is possible but not convincing and the need for a bet-

ter explanation is especially pressing, since some studies provided data which are extraor-

dinary and cannot be ignored. Additionally, 8 out of 17 studies were able to provide evi-

dence that M. avium subsp. paratuberculosis is a necessary and sufficient condition of

Crohn's disease. In this context, 17 of 17 studies provided data which are consistent with

the conclusion that there is a significant cause and effect relationship between M. avium

subsp. paratuberculosis and Crohn's disease. Besides of the very limited number of cases

and controls studied, the present study confirms previous reports of the association be-

tween M. avium subsp. paratuberculosis and Crohn's disease. More particularly, according

to Zamani et al. [39] Mycobacterium avium subsp. paratuberculosis is a necessary condi-

tion of Crohn's disease and equally a sufficient condition of Crohn's disease while the

cause effect relationship between Mycobacterium avium subsp. paratuberculosis and

Crohn's disease is highly significant. In point of fact, i. e. how could Zamani et al. [39]

provide such an impressive evidence of the relationship between Mycobacterium avium

subsp. paratuberculosis and Crohn's disease? The data of Zamani et al. [39] are viewed by


Ilija Barukčić


the Table 9.

Table 9. The data of Zamani et al. [39].

Crohn’s disease (CD)

Yes No Total

Mycobacterium avium Yes 18 6 24

subsp. paratuberculosis (MAP) No 10 56 66

Total 28 62 90

Results:

p(UW)= 0,8889

p(UW)= 0,93333333

X²(UW) = 1,3674

X²(UW) = 1,26041667

k(U,W) = 0,5717

p val (k)= 6E-08

Altogether, the results of this study support the hypothesis that M. avium subsp. paratu-

berculosis is the cause of Crohn's disease which implicates the necessity of an effective

antibacterial treatment supported by a plant based [90] lactose free diet of this mycobacte-

rial intestinal infection. Once questions raised by methodological inconsistencies and their

difficulty are acknowledged, a treatment of Crohn's disease patients with appropriate anti-

biotics for sufficient duration able kill Mycobacterium avium ssp. paratuberculosis (MAP)

is worth being considered. Effective treatment of a mycobacterial infection can be diffi-

cult, due to the structure of the mycobacterial cell wall. In principle, the mycobacterial cell

wall can hinder the entry of drugs and is able to make many antibiotics ineffective. Myco-

bacterium avium ssp. paratuberculosis (MAP) as the etiologic agent of cattle's paratuber-

culosis (Johne's disease) lacks its cell wall in humans [91] while more than 30 strains [92]

of MAP have been identified. Cell wall deficient bacterial organisms may lack important

cell wall antigens more than cell wall competent bacterial organisms, and so are less visi-

ble to host immune surveillance and theoretically may survive better than other organism.

The human immune system shaped through evolution by the necessity of discriminating

non-self pathogens from self tissues and extremely important for the survival of a multi-

cellular organisms uses especially antigens to determine which cells are resident and

which are foreign. In the following, antibodies can recognize an antigen and lock onto it

without being capable of destroying it without help. Mycobacterium avium ssp. paratu-

berculosis appears to avoid recognition by the immune system and makes the triggering of

immune responses against itself to a very great extent ineffective. Parameters like

C-reactive protein, Erythrocyte sedimentation rate, (Procalcitonin (PCT)), IgG-, IgM, IgA

antibodies against Mycobacterium avium ssp. paratuberculosis may provide only a weak

and approximate picture of the extent of inflammation. In particular, using antibiotic drugs

which aim to target the cell wall of Mycobacterium avium ssp. paratuberculosis may lead

to complications, the rise of multidrug resistance bacteria and inhibiting of normal flora.

At the end, such a strategy could be inefficient for treatment of Crohn’s disease patients.


Ilija Barukčić


The evidence presented in this study based on a systematic review of other studies justify

the assumption to treat Crohn’s disease with antibiotic. The following or similar Phase I

(Table 10) and Phase II (Table 11) drug regimen for the treatment of Crohn’s disease pa-

tients is still not established. Whether does it makes sense to stop the therapy with Rifaxi-

min 550 mg b.i.d. (i. e. or Metronidazol 400 mg b.i.d. and Ciprofloxacin 500 mg b.i.d.)

which is used to control intestinal microbiome dysbiosis and Predinsolon 60 mg q.d. (in

this context used to control clinical symptoms) after two months of therapy is another

point which is not secured. Diagnostic (or therapeutic) colonoscopy if necessary with the

support of a feeding / gastroduodenal tube is needed for therapeutic monitoring and to re-

duce the microflora of the gut mechanically. The therapy with drugs free of lactose (a di-

saccharide composed of galactose and glucose) like Clarithromycin 500 mg b. i. d.,

Rifabutin 450 mg q. d. and Colofazimine 50 mg q. d. should continue for at least further 4

months or longer until a complete remission is achieved. Although shortening the duration

of therapy is a desirable target, the use of the standard 4-month rifabutin-containing regi-

men or longer is worth being considered.

Table 10. Phase I. The starting 2 months antibiotic regimen to treat MAP in Crohn’s disease.

Week 1 2 3 4 5 6 7 8

Predinsolon 60 mg q.d. (x) (x) (x) (x)

Rifaximin 550 mg b.i.d. x x x x

Probiotics / 25 OH Vitamin D3 … x x x x

(diagnostic or therapeutic) colonoscopy x x x x

Furthermore:

Colofazimine 50 mg q.d. x x x x x x x x

Clarithromycin 500 mg b.i.d. x x x x x x x x

Rifabutin 450 mg q.d. x x x x x x x x

Safety evaluation, risk control and therapeutic monitoring should consider at least prame-

ters like C-reactive protein, Erythrocyte sedimentation rate, (Procalcitonin (PCT)), IgG-,

IgM, IgA antibodies against Mycobacterium avium ssp. paratuberculosis, faecal calprotec-

tin and of course other too. The novelty of this study is focused on the causal relationship

between Crohn’s disease and Mycobacterium avium ssp. paratuberculosis. Reports about

the relationship between Crohn's disease (CD) and cell wall-deficient (CWD) forms of

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) are still contro-

versial and even this study must acknowledge that a causal link between Mycobacterium

avium subspecies paratuberculosis (MAP) and Crohn's disease has not been established

beyond any reasonable doubt, arguments to the contrary equally have to be considered.

Besides of the difficulties mentioned growing evidence determined by the studies present-

ed suggests that Mycobacterium avium subspecies paratuberculosis is a necessary and

/ or sufficient condition of Crohn's disease.


Ilija Barukčić


Table 11. Phase II. The 4 months and longer antibiotic regimen to treat MAP in Crohn’s disease.

Month 3 4 5 6 7 8 9 10

Colofazimine 50 mg q.d. x x x x x x x …

Clarithromycin 500 mg b.i.d. x x x x x x x …

Rifabutin 450 mg q.d. x x x x x x x …

(diagnostic or therapeutic) colonoscopy x (x) (x) x (x) (x) x …

Probiotics / 25 OH Vitamin D3 … x x x

At the same time, all studies provided evidence of a significant cause effect relationship

between and Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn's dis-

ease. This article provides a review of recent PCR DNA based works on the relationship

between and Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn's dis-

ease and invites us to consider the following inescapable conclusion.

5. Conclusion

Mycobacterium avium subspecies paratuberculosis (MAP) is the cause of Crohn's disease.

Acknowledgements

None.

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